Urinary tract infections (UTIs) are among the most frequent bacterial infections afflicting humans and have a high degree of recurrence. With rising resistance to front-line antibiotics, there is a pressing need for the development of targeted strategies aimed at preventing and/or treating UTIs. Uropathogenic Escherichia coli (UPEC) account for the majority of UTIs and can form robust extracellular biofilms, as well as biofilm- like intracellular bacterial communities (IBCs). Among the factors impacting IBC formation is the QseC sensor kinase, the disruption of which diminishes IBC formation and attenuates UPEC virulence. We have shown that QseC is required for the de-phosphorylation and deactivation of its cognate response regulator QseB, which becomes constitutively activated in the absence of QseC. Constitutively active QseB leads to the dysregulation of >500 genes, interferes with core metabolic processes and downregulates virulence gene expression. Investigating the source of QseB phosphorylation in the absence of QseC identified the PmrAB two-component system (TCS) as the primary QseB activator. We went on to show that in wild-type UPEC, signal reception by PmrB can also activate the non-cognate partner QseB, indicating a potential overlap between QseBC and PmrAB signaling. These results lead to two hypotheses: 1) There is robust cross-talk between non-cognate TCS partners that is of physiological significance during UPEC pathogenesis and, 2) We can target QseC function as a means to attenuate virulence. This proposal will engage fundamental questions about the regulatory interactions between QseBC and PmrAB and their role in UTI pathogenesis, and endeavor to harness this information to treat/prevent UTIs. Aims 1 and 2 will elucidate the molecular mechanisms underlying the cross-talk between UPEC QseBC and PmrAB. Aim 3 will probe the role of QseBC-PmrAB interactions during infection and will identify compounds that target the QseC phosphatase function, and/or bias cross-talk between QseB and PmrB. We have developed an extensive array of molecular tools that we are using in our studies, including non-polar deletion mutants, as well as inactive or constitutively active point mutants. Combined with the well-established murine model of UTI we are using, our microscopy capabilities to track infection in the bladder, and the impressive facilities and resources of Vanderbilt University, we are confident that we will harness information that will elucidate the QseBC-PmrAB molecular interplay within and outside the host. Our long-term goal is to leverage the outcomes of the herein proposed studies to develop better therapies against UPEC infection.